The flavivirus family includes several clinically important animal viruses, including Dengue, West Nile, Japanese encephalitis, yellow fever, and tick-borne encephalitis viruses. Dengue is one of the most serious infectious diseases globally. There are about 300 million cases every year, with over 500,000 cases of potentially fatal Dengue hemorrhagic fever. Dengue virus (DENV) puts nearly 2.5 billion people at risk of infection in tropical and subtropical countries. Similarly, West Nile virus (WNV) has caused thousands of human infections in North America, besides infecting people on other continents. WNV infection can lead to serious illnesses in humans, resulting in encephalitis and death. Neither a prophylactic vaccine nor antiviral therapies are available for both WNV and DENV. The development of either a vaccine or an antiviral drug requires detailed knowledge of the viral life cycle.
The spherical, approximately 50 nm diameter dengue virion contains an inner nucleocapsid made up of the plus-sense RNA genome and multiple copies of the viral core protein and is sheathed in a lipid bilayer derived from the host cell. The virion membrane is coated with 180 copies of the envelope (E) protein that form 90 homodimers arranged in a tight herringbone structure. Following attachment to the cell surface, the dengue virion is internalized via a clathrin-dependent process. The viral nucleocapsid escapes from the endosomal compartment via a pore created upon fusion of the viral and endosomal membranes. This process of viral fusion is catalyzed by the E protein and is triggered by acidic pH. The viral genome is translated to produce a single polyprotein that is post-translationally processed by cellular and viral proteases to produce the ten DENV proteins. Replication of the viral genome is catalyzed by the viral RNA-dependent RNA polymerase, NS5, and occurs in membrane-associated complexes in the perinuclear region. Upon encapsidation of the viral genomic RNA by the core protein, the nucleocapsids bud into the endoplasmic reticulum lumen, a process that leads to their acquisition of a lipid membrane and association with 180 heterodimers of the viral E and prM proteins organized as quasi-trimers arranged perpendicularly to the virion surface. prM functions as a chaperone protein to prevent premature triggering of the E protein on immature viral particles within the acidic environment of the secretory pathway. Mature viral particles are produced upon cleavage of prM by furin and rearrangement of E into homodimers during exocytosis. The DENV infectious cycle is known to occur on the timescale of several hours, with the release of progeny viral particles commencing at 12 to 24 hours following infection, depending upon the virus strain and cell-type.
Based on its tertiary structure, the DENV E protein is a class II viral fusion protein. Two transmembrane domains anchor the E protein in the viral membrane and are linked to three globular domains (domains I, II, and III) via a short “stem” region and membrane proximal helix-loop-helix. Domain I is a β-barrel forming the core of the protein monomer. The immunoglobulin-like domain III acts as the putative receptor-binding domain and is the major site of neutralizing antibody epitopes. The fusion loop located at the tip of the “finger-like” domain II contacts domain III of the dimer partner. The large-scale structural changes triggered by the acidification of the endosomal compartment catalyze fusion of the viral and target membranes.
Viruses can be interrogated using chemical tools; for example, small molecules or RNAi may be used to identify host factors or pathways integral for viral replication or viral entry. Specifically, host proteins and enzymes that may be important for viral replication may be probed by measuring the effect on yield of viral particles in the presence of known inhibitors of a specific protein or enzyme. In addition, small molecules may inhibit viral entry by interacting with viral proteins, such as envelope protein E.
In general, there exists a need for antiviral therapies that are not detrimental to host cell viability.